1. Field of the Invention
This invention relates generally to recombinant DNA techniques and to the expression of mammalian polypeptides in genetically engineered eukaryotic cells. Specifically, the invention relates to preferred plasmid constructs and methods that increase levels of expression of a cloned gene product. These preferred plasmids have selectable and nonselected gene cassettes adjacent to each other and oriented in the opposite direction for transcription. Such an orientation enhances overall levels of expression for the nonselected gene. Further, this invention relates to gene products expressed at these very high levels by the herein described method, and to the eukaryotic cells derived thereby. 2. Background and Prior Art
The ability to produce cloned gene products in eukaxyotic cell culture is often desirable, particularly when the cloned gene encodes a eukaryotic polypeptide, since prokaryotes lack a number of elements contained in eukaryotic cells. Such elements include the eukaryotic signals for transport, modification and glycosylation. Proper eukaryotic post-translational modification is often necessary for normal function of the final recovered protein. The eukaryotic gene product is most similar to or the same as the natural gene product when the cloned gene is expressed in a eukaryotic cell.
Early transfection experiments of Wigler, M. et al (Cell 11: 223-232 1977) were effective but inefficient. Since Axel first described cotransformation to increase the efficiency of transfecting foreign DNA into host cells (U.S. Pat. No. 4,399,216 and Wigler et al. Cell 16: 777-788, 1979), it has become common to have a drug resistance selection marker and a non-selectable gene of interest on the same plasmid as separate transcription units, each with their own promoter (Girli, I., Jouanneau, J. and Yabiv, M., 1983, Virology 127: 385-396 and Southern, P. J., Berg, P., 1982, J. Mol. Appl. Genet. 1:327-341). Expression levels of the non-selectable gene vary greatly in the individual transfected clones arising from experiment to experiment.
The current methodology usually includes first selecting a population of cells containing the transfected plasmid on the basis of drug resistance. This population is then screened for the introduction of a non-selectable gene, either by assaying for the non-selectable gene product, or for the presence of the genetic material comprising the coding sequence of the non-selectable gene. This method allows for the stable introduction into cultured mammalian cells of any cloned gene, and the systematic isolation of such cells.
A major issue in this area of research is the need to develop systems which can provide high levels of expression of the gene of interest in a reliable and reproducible way. Several methods to improve gene expression have been used successfully.
Expression of one gene in a multi-gene plasmid may be affected by other gene in the plasmid. Roginski et al. (Cell 35: 149-155, 1983) constructed a plasmid containing three transcription units. Clones were selected using the TK selection system and the expression of the non-selectable globin gene was analyzed. This group found coordinate expression of all three genes in 8 of the 10 clones examined. However, two other groups (Emiman, M. and Temin, H. M., Cell 39, 459-467, 1984 and; Proudfoot, N. J., Nature 322:562-565, 1986) have described the effect of transcriptional interference on adjacent cistrons. In these tWo examples, the expression of one gene had a negative effect on the expression of the adjacent gene.
Gene amplification is proven to be a method frequently used to increase expression levels. Gene amplification is often induced by exposure of sensitive cells to stepwise increases in antifolates, such as methotrexate (MTX), in the growth medium. This amplification can yield cells which are resistant to high levels of MTX and have increased levels of production of other associated genes as well (European Patent Application Nos. 0117059, 00117060 and Kaufman et al. (1985) Mol. Cell. Biol. 5:1750-1759, 1985).
However, gene amplification has several drawbacks including the instability of resultant clones, and the need to grow cultures in presence of high level of carcinogens (Kaufman and Sharp, J. Mol. Biol., 159:601-621, 1982).
In general, increased production of foreign proteins in a host through gene amplification techniques has been primarily limited to the use of mutant hosts. This is often undesirable for a number of reasons, most notably, that the mutant cell host most suitable for production of the foreign protein is unavailable. The expression of genes into fully active proteins commonly requires a specific cell type, which may not be available with the necessary mutation.